Use of antisecretory factors (af) for optimizing cellular uptake

ABSTRACT

The present invention relates to the use of an antisecretory factor (AF) protein, peptide, derivative, homologue, and/or fragment thereof, having equivalent functional activity, and/or a pharmaceutically active salt thereof, for optimizing delivery and cellular uptake of a pharmaceutical substance and/or formulation, or a gene delivery. Typically, said pharmaceutical substance and/or formulation comprises an anticancer drug, radiation therapy, an antibiotic substance, an antiviral substance or a drug targeting posttraumatic injury, neurodegeneration, a parasite, or an inflammatory condition.

FIELD OF INVENTION

The present invention relates to the use of an antisecretory factor (AF)protein, peptide, derivative, homologue, and/or fragment thereof, havingequivalent functional activity, and/or a pharmaceutically active saltthereof, for optimizing delivery and cellular uptake of a pharmaceuticalsubstance and/or formulation, or a gene delivery. Typically, saidpharmaceutical substance and/or formulation comprises an anticancerdrug, radiation therapy, an antibiotic substance, an antiviral substanceor a drug targeting sequels of posttraumatic brain injuries,neurodegeneration, a parasite, or an inflammatory condition.

In general, the present invention relates to the surprising insight thatantisecretory factor (AF) actively influences the equilibrium betweenphosphorylated and dephosphorylated states of a vast number offunctional proteins and that it in particular can intervene in thebiological activation of transmembrane proteins, such as Na⁺-K⁺-2Cl⁻co-transporter (NKCC1), by interacting with CAP/ponsin (c-Cbl associatedprotein) and FAK (focal adhesion kinase). AF can thus effectivelyregulate and/or normalize abnormal activity of said ion channel inperturbed and/or pathological cells, thereby effectively normalizing theintracellular pressure in the pathological cell, potentially allowing animproved cellular uptake of a pharmaceutical substance, such as a drugused in e.g. cancer, inflammation, or trauma therapy, or a nucleic acidsequence used for gene delivery.

The present invention further of course also relates to the use of theabove described surprising insight that antisecretory factor (AF)protein as well as a peptide derived thereof comprising the essentialconsensus sequence of AF can intervene in the biological activation oftransmembrane proteins and/or in particular co-transporters, such asNKCC1, through CAP/ponsin and FAK in a broad variety of methods forimproved drug design, for screening for and/or evaluating potential AFinhibitory and/or enhancing substances, and for evaluating efficacyand/or verifying functional activity of a new or known antisecretoryfactor (AF) protein, peptide, derivative, homologue, and/or fragmentthereof, having equivalent functional activity, and/or apharmaceutically active salt thereof.

In another aspect, the newfound insight of AF's biological cellularfunction further enables the design of a new and reliable diagnosticand/or prognostic tool for monitoring and/or verifying and/or enhancingthe therapeutic control of a cancer and/or abnormal tissue growth oractivity in a subject suffering e.g. from cancer.

BACKGROUND OF THE INVENTION

Defective regulation of cell secretion underlies the clinicalmanifestations of a number of important human diseases ranging fromcystic fibrosis to secretory diarrhea and brain edema.

The regulation of metazoic cells' dimensions and internal milieu is avery high priority matter for their normal function, multiplication andsurvival. The cytoskeleton, formed by the fairly static microtubules andintermediate filaments, and the dynamic actin filament network act assensors of cells' shape, dimensions, three dimensional shape andmechanical load. Excessive inflow of water and ions increase a cell'sdimensions, e.g., as occurring if the cell is exposed to a hypotonicextracellular environment. In contrast, a hypertonic extracellularmilieu renders the cell to shrink. Any divergence from the normal statusof a cell is immediately and forcefully counteracted as a cell, for thesake of its survival and optimal function, is giving its highestpriority to maintain a “normal” status. Thereby, the actin filaments andassociated myosin 1 form a sensing system alarming on any divergencefrom the normal conditions. The actin filaments are attached to lipidrafts and caveolae, as well as to junctional complexes anddesmosomes/hemidesmosomes and to other constituents of the cytoskeleton,microtubules and intermediate filaments, thereby being able to read thecondition prevalent in a cell. The actin filament system is linked toprotein complexes at the cytoplasmic face of lipid rafts and caveolae,which arrangement enables the actin filaments to monitor the effectors'parts of the cell. Thereby the signals emitted by the actin filamentsinduce counteractions, enabling the cell to maintain its normal status.These actin linked proteins are e.g. galectin, filamin and flotillin,and they often form oligomers. The flotillin-1 and flotillin-2 formoligomers, mostly tetramers, which are anchoring lipid rafts to theactin filament network. Flotillin-1 binds with very high affinity to theAF protein as well as to the AF-derived peptide AF-16, while flotillin-2is firmly linking actin filaments. The level of flotillin-1 is rapidlyadjusted by degradation of free flotillin-1, rendering dynamics to thesystem. It is known that flotillins monitor certain kinases andphosphatases, which in turn regulate the activity of transmembraneproteins, e.g. ion pumps and G-protein linked systems such as NKCC's,the activity of which are further linked to the levels and activities ofCAP/ponsin and FAK.

Optimization of drug and gene delivery is a topic of great interest. Theoptimization can be achieved via site-specific and targeted delivery,controlled drug release, and by finding ways to deliver higherconcentrations of a drug into tissues of interest despite variousbarriers. Targeted delivery can serve to lower the required drug doseand minimize toxic side effects, which is e.g. crucial for the successof cancer treatment by immunotherapy, chemotherapy and/or radiation.Controlled release of drugs can be advantageous in the management ofchronic diseases such as trauma-related conditions, neurodegenerativediseases, diabetes and hypertension.

Chemotherapy and immunotherapy are therapeutic approaches of majorimportance for the treatment of both localized and metastasized tumors.Since anticancer drugs are neither specific nor targeted to the cancercells, improved delivery of anticancer drugs to tumor tissues in humansappears to be a reasonable, beneficial and achievable challenge.Scientists are working to increase the availability of drug for tumoruptake by 1) delaying the release preparations for long-lasting actions;2) using liposome-entrapped drugs for prolonged effect or reducedtoxicity; 3) administrating inert, non-toxic prodrugs for specificactivation at the tumor site; 4) delivering antibody-mediated drugs; or5) conjugating site-specific carriers to direct the drug to the tumortarget. The latter depends heavily on pharmacokinetic investigations.Some success has been achieved in enhancing the efficacy and reducingthe toxicity of drugs.

What is more, it is generally known in the field that the response oftumor to various anticancer drugs is tumor-size dependent in manyaspects. In general, problems stem partly from the fact that the entiretumor cell populations do not respond equally to a certain treatment. Asa result of recent progress in cancer biology, it has become evidentthat cellular heterogeneity of the tumor underlies the difficulties oftreating primary and metastatic tumors with chemotherapy. Moreover, astumors grow, marked diversity develops on the tissue level as well. Anuneven distribution with an increase of areas of lower growth fractionand of poorer drug delivery is more distinct in larger tumors.Heterogeneous distribution and low levels of tumor blood flow areconsidered to be causally related to the heterogeneous nature of tumortissue. Considering the lack of evidence of a lymphatic system withinthe tumor, increased interstitial fluid pressure may be a natural resultthat further impedes blood flow in the tumor.

The efficacy in cancer treatment of novel therapeutic agents such asmonoclonal antibodies, cytokines and effector cells has been limited bytheir inability to reach their target in vivo in adequate quantities.Molecular and cellular biology of neoplastic cells alone has failed toexplain the non-uniform uptake of these agents. This is not surprising,since a solid tumor in vivo is not just a collection of cancer cells. Infact, it consists of two extracellular compartments: vascular andinterstitial. Since no blood-borne molecule or cell can reach cancercells without passing through these compartments, the vascular andinterstitial physiology of tumors has received considerable attention inrecent years. Three physiological factors responsible for the poorlocalization of macromolecules in tumors have been identified: (i)heterogeneous blood supply, (ii) elevated interstitial pressure, and(iii) long transport distances. The first factor limits the delivery ofblood-borne agents to well-perfused regions of a tumor; the secondfactor reduces extravasations of fluid, nutritients, oxygen andmacromolecules in the high interstitial pressure regions and also leadsto an experimentally verifiable, radial outward convection in the tumorperiphery which opposes the inward diffusion. The third factor increasesthe time required for slowly moving macromolecules, nutritients, oroxygen to reach distant regions of a tumor. Binding of any molecule toe.g. an antigen further lowers the effective diffusion rate by reducingthe concentration of mobile molecules. Although the effector cells arecapable of active migration, peculiarities of the tumor vasculature andinterstitium may also be responsible for poor delivery of lymphokineactivated killer cells and tumor infiltrating immuno active cells insolid tumors. Due to micro- and macroscopic heterogeneities in tumors,the relative magnitude of each of these physiological barriers wouldvary from one location to another and from one day to the next in thesame tumor, and from one tumor to another. If genetically engineeredmacromolecules and effector cells, as well as low molecular weightcytotoxic agents, are to fulfill their clinical promise, strategies mustbe developed to overcome or exploit these barriers.

Solid tumors, enclosed by a capsule and sometimes divided by septa,often develop high interstitial fluid pressure (IFP) as a result ofincreased fluid leakage and impaired blood circulation and lymphaticdrainage, as well as changes in the extracellular matrix composition andelasticity. This means that the arteriolar blood pressure at manyoccasions causes the IFP to reach high levels. Also swelling of tumorcells contributes to the raised IFP. Raised interstitial fluid pressureforms a barrier to drug delivery and hence, resistance to therapy.

A cell undergoes genetic and epigenetic changes during its transition tomalignancy. Malignant transformation is also accompanied by aprogressive loss of tissue homeostasis and perturbations in tissuearchitecture that ultimately culminates in tumor cell invasion of theparenchyma and metastatic spread to distant tissue and organ sites.Increasingly, cancer biologists have begun to recognize that a criticalcomponent of this transformation journey involves marked alterations inthe mechanical phenotype of the cell and its surroundingmicroenvironment. These include modifications in cell and tissuestructure, adaptive force-induced changes in the environment, alteredprocessing of micromechanical cues encoded in the extracellular matrix(ECM), and cell-directed remodeling of the extracellular stroma. Solidtumors are commonly stiffer than normal tissue, and tumors have alteredintegrins.

Growing evidence indicates that critical steps in cancer progressionsuch as cell adhesion, migration, and cell cycle progression are inparts regulated by the composition and organization of themicroenvironment. The adhesion of cancer cells to components of themicroenvironment and the forces transmitted to the cells via the actinnetwork and the signaling complexes organized at focal adhesions, lipidrafts and caveolae, allow cancer cells to sense the local topography ofthe extracellular matrix and respond efficiently to growth and migrationpromoting cues.

The cytoskeleton, including its actin network, is known to be of crucialimportance for the structure and function of normal, inflammatory andneoplastic cells. At e.g. a brain trauma, the cytoskeleton isextensively deranged at locations and to an extent varying with theapplied forces. The actin filaments are as well disintegrating atencephalitis (Jennische et al., 2008) and at cholera toxin induceddiarrhea (Hansson et al., 1984). Thus, the crucial roles of thecytoskeleton for the maintenance of normal dimensions of cells and forthe emergence of dysfunctions are established.

Much attention has focused on the role of membrane chloride (Cl⁻)channels in the maintenance of normal cell functions, emerging evidencehighlights the importance of the Na⁺-K⁺2Cl⁻ co-transporter (NKCC) as anindependent regulatory site that may determine the overall rate of cellsecretion. The co-transporter NKCC1 is expressed in virtually allmammalian cells, where it plays a more generalized role in cell volumehomeostasis, cell ionic composition, and, possibly, the control of cellgrowth. Emerging molecular evidence indicates that NKCC1 function isregulated in the short and long term at the level of proteinphosphorylation, membrane targeting, and gene expression (Mathews,2002). Thus, an improved understanding of the interactions between thecytoskeleton, flotillin oligomers, lipid rafts and effectors such asNKCC1 has lead the present inventors to new therapeutic approaches tocancer and to neurodegeneration, as well as to the treatment of a rangeof clinical conditions in which the cell dimensions and ion compositionare disturbed.

The Na—K—Cl co-transporters are a class of membrane proteins thattransport Na⁺, K⁺, and Cl⁻ ions into and out of a wide variety ofepithelial and non-epithelial cells. The transport process mediated byNa—K—Cl co-transporters is characterized by electro neutrality (almostalways with stochiometry of 1 Na:1K:2Cl) and inhibition by the “loop”diuretics such as bumetanide, benzmetanide, and furosemide. Presently,two distinct Na—K—Cl co-transporter isoforms have been identified bycDNA cloning and expression; genes encoding these two isoforms arelocated on different chromosomes and their gene products shareapproximately 60% amino acid sequence identity.

The NKCC1 (CCC1, BSC2) isoform is present in a wide variety of tissues.Most normal epithelial cells containing NKCC1 are secretory epitheliawith the Na—K—Cl co-transporter localized to the basolateral membrane.By contrast, NKCC2 (CCC2, BSC1) is found only in the kidney, localizedto the apical membrane of the epithelial cells of the thick ascendinglimb of Henle's loop and of the macula densa. Mutations in the NKCC2gene result in Bartter's syndrome, an inherited disease characterized byhypo potassium metabolic alkalosis, hypercalciuria, salt wasting, andvolume depletion. The two Na—K—Cl co-transporter isoforms are also partof a superfamily of cation-chloride co-transporters, which includeselectroneutral K—Cl and Na—Cl co-transporters. Cancer cells, whichmostly are apolar, do express high levels of NKCC, resulting in that thetumor cells in fact are swollen, i.e. having increased dimensions. Tumorcells have less precisely regulated ion pump and water channel systems,but still show a strong tendency to maintain their internal homeostasis.That means that the increased dimensions of tumor cells, enclosed by acapsule, contribute to the raise of the IFP common in solid tumors.

Na—K—Cl co-transporter activity is affected by a large variety ofhormonal stimuli as well as by changes in cell volume. In many tissuesthis regulation (particularly of the NKCC1 isoform) is regulated by thebalance between phosphorylation and dephosphorylation of regulatorysystems, controlling the ion pumps prevalent in the lipid rafts throughthe specific protein kinases or phosphatases. (Haas, 1998)

Cell shrinkage-induced activation of NKCC involves an interactionbetween the cytoskeleton and protein phosphorylation events via PKC andmyosin light chain kinase (MLCK). Osmotic control of Cl— secretionacross the epithelium includes: (i) hyperosmotic shrinkage activation ofNKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC byhypotonic cell swelling and a protein phosphatase, and (iii) a proteintyrosine kinase acting on the focal adhesion kinase (FAK) to set levelsof NKCC activity. The CAP component is interposed as well and takesparts in the step wise regulation of the extent of phosphorylation ofNKCC, which determines its function within the lipid rafts (Hoffmann2007).

At the electron microscopic level, a unique combination of integrin β1,the phosphorylated form of FAK at tyrosine 407 (pY407) and Na(+), K(+),2Cl(−) co-transporter (NKCC1) were all co-localized only on thebasolateral membrane in normal cells. The three proteins were alsoco-immunoprecipitated with each other in isotonic conditions, suggestingan osmosensing complex involving the three proteins. Only FAK pY407 wassensitive to hypotonic shock and became dephosphorylated with hypotonicshock, while FAK pY576 in the apical membrane and pY861 in cell-celladhesions were insensitive to hypotonicity. It has been reported thatchloride cells respond to hypotonic shock using integrin β1 as anosmosensor that is connected to dephosphorylation of FAK pY407 whichleads to NKCC1 deactivation in the basolateral membrane and theinhibition of NaCl secretion by these epithelial cells (Marshall, 2008).Again, tumor cells commonly are apolar and thus the ion pumps in thelipid rafts are localized all along the cell surface, as apical andbasolateral areas are not prevalent. Thus, the same kind of ion pump isprevalent in normal cells as in cancer cells and in either casesimilarly monitored, albeit their localizations differ.

Integrins are cell surface receptors which, in part, mediate theadhesion of cells to the extracellular matrix. In addition to providingmolecular “glue” essential for tissue organization and survival,integrins serve as dynamic signaling molecules. Integrins allow normal,non-transformed cells to sense that they are adhered to theextracellular matrix, thus providing a cell survival signal. This signalallows cells to proliferate in the presence of growth factors and insome instances prevents apoptosis. Integrins also mediate cell migrationas it occurs in normal processes, such as angiogenesis, wound healing,repair of damage, monitor immune system function, and development.Aberrances in the expression and function of integrins contribute tomany diseases and disorders, including cancer.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that isoverexpressed in a variety of cancers and plays an important role incell adhesion, migration, and anchorage-dependent growth (Tilgham, 2007)

Focal adhesion kinase (FAK), prevalent in practically all normal cells,is overexpressed in invasive and metastatic colon, breast, thyroid, andprostate cancers. Enhanced FAK immunostaining is detected in smallpopulations of preinvasive (carcinoma in situ) oral cancers and in largepopulations of cells in invasive oral cancers. It has been hypothesizedthat FAK is probably not a classical oncogene but may be involved in theprogression of cancer to invasion and metastasis. It is furtherhypothesized that overexpression of FAK in subpopulations of tumor cellsleads to populations of cells with a high propensity toward invasion andmetastasis (Kornberg, 1998).

Focal adhesion kinase (FAK) localizes to cellular focal adhesions orcell contacts within the extracellular matrix. FAK is activated by avariety of cell surface receptors and transmits signals to a range oftargets. FAK participates in growth factor receptor-mediated signalingpathways and plays essential roles in cell survival, proliferation,migration, and invasion.

Overexpression of FAK is widely observed in numerous tumor types, and isused as a marker for invasion and metastasis. FAK could betherapeutically targeted at various levels, such as at the level of FAKgene transcription by regulating its transcription factor(s) with siRNA,at the FAK mRNA level with FAK siRNA, or at the protein level. At theprotein level, FAK's localization to lipid rafts in focal adhesionscould be disrupted by expression of dominant-negative FAK-RelatedNon-Kinase or its focal adhesion targeting domain, and its kinaseactivity could be inhibited by FIP200, the FAK kinase domain-interactingprotein and kinase-activity inhibitor. In recent years, research hasbeen focused on developing small molecule inhibitors against FAKtranscription and activation, to provide additional approaches forpotential tumor therapies (Lis. 2008).

Another substrate involved in the phosphorylation of intracellularsubstrates regulating the transduction and control of signalsdetermining the level of activity of ion pumps is CAP, which is anadapter protein for the Cbl proto-oncogene product. CAP is acting as alink in the signaling pathway at the cytoplasmic leaflet of lipid raftsbetween flotillin and FAK. CAP is also known under the name ponsin. Thename reflects that this factor originally was isolated and identified inover-expressing tumors and therefore named proto-oncogene product, buthas subsequently been disclosed to be prevalent as well in normal cells.

Antisecretory factor is a 41 kDa protein that originally was describedto provide protection against diarrhea diseases and intestinalinflammation (for a review, see Lange and Lönnroth, 2001). Theantisecretory factor (AF) protein has been sequenced and its cDNAcloned. The antisecretory activity seems to be mainly exerted by apeptide located between the amino acid positions 35 and 50 on theantisecretory factor (AF) protein sequence and comprising at least 4-16,such as 4, 6, 8 or 16 amino acids of the consensus sequence.Immunochemical and immunohistochemical investigations have revealed thatthe antisecretory factor (AF) protein is present and may also besynthesized by most tissues and organs in a body. Synthetic peptides,comprising the antidiarrhoeic sequence, have prior been characterized(WO 97/08202; WO 05/030246). Antisecretory factor (AF) proteins andpeptides have previously been disclosed to normalize pathological fluidtransport and/or inflammatory reactions, such as in the intestine andthe choroid plexus in the central nervous system after challenge withthe cholera toxin (WO 97/08202). Food and feed with the capacity toeither induce endogenous synthesis of AF or uptake of added AF havetherefore been suggested to be useful for the treatment of edema,diarrhea, dehydration and inflammation in WO 97/08202. WO 98/21978discloses the use of products having enzymatic activity for theproduction of a food that induces the formation of antisecretory factor(AF) proteins. WO 00/038535 further discloses the food products enrichedin antisecretory factor (AF) proteins as such.

Antisecretory factor (AF) proteins and fragments thereof have also beenshown to improve the repair of nervous tissue, and proliferation,apoptosis, differentiation, and/or migration of stem and progenitorcells and cells derived thereof in the treatment of conditionsassociated with loss and/or gain of cells (WO 05/030246) and to beequally effective in the treatment and/or prevention of intraocularhypertension (WO 07/126,364), as for the treatment and/or prevention ofcompartment syndrome (WO 07/126,363).

What is more, the present inventors recently showed that antisecretoryfactors were able to monitor and/or beneficially affect the structure,distribution and multiple functions of lipid rafts, receptors and/orcaveolae in membranes and could thus be employed for the treatmentand/or prevention of structural disorganization and dysfunction of lipidrafts and/or caveolae in cell membranes (WO 07/126,365).

Surprisingly, the present inventors have now been able to prove that thesame antisecretory factors can intervene in the above describedbiological activation of transmembrane proteins, e.g. NKCC1 through FAKand CAP, and can thus directly regulate the pathological activity of theion channel in pathological and/or perturbed cells, effectivelynormalizing the intracellular pressure and transmembrane proteinfunction in said cell, and thus allowing an improved uptake of drugsused in e.g. cancer therapy.

SUMMARY OF THE PRESENT INVENTION

The present invention relates to the use of a pharmaceutical compositioncomprising an antisecretory factor (AF) protein, a homologue,derivative, peptide and/or fragment thereof, having antisecretory and/orequivalent functional and/or analogue activity, preferably beingselected from an antisecretory factor (AF) protein consisting of asequence according to the following formula

X1-V-C-X2-X3-K-X4-R-X5,

wherein X1 is I, amino acids 1-35 of SEQ ID NO 6, or is absent, X2 is H,R or K, X3 is S or L, X4 is T or A, X5 is amino acids 43-46, 43-51,43-80 or 43-163 of SEQ ID NO 6, or is absent, or a modification thereofnot altering the function of the polypeptide, or a pharmaceuticallyactive salt thereof, for the manufacture of a pharmaceutical compositionfor optimizing delivery and/or cellular uptake of a second or furtherpharmaceutical substance and/or formulation.

Typically, said second or further pharmaceutical substance and/orformulation comprises an anticancer drug, radiation therapy, antibioticsubstance, antiviral substance, and/or a drug targeting posttraumaticinjury, neurodegeneration, a parasite, or an inflammatory condition.

The present invention in another equally preferred aspect also relatesto the use of an antisecretory factor (AF) protein, peptide, derivative,homologue, and/or fragment thereof, having equivalent functionalactivity, and/or a pharmaceutically active salt thereof, for optimizingdelivery and cellular uptake of a gene delivery.

In general, the present invention relates to the surprising insight thatantisecretory factor (AF) in particular can intervene in the biologicalactivation of transmembrane proteins, such as NKCC1 by interacting withCAP/ponsin (c-Cbl associated protein) and/or FAK (focal adhesionkinase).

AF has prior been known to co-localize with flotillin 1 and 2, a proteinthat is anchoraged in the lipid raft of the cell membrane and whichbinds to actin and to CAP, which in turn binds to the SHP-2/PTPD1 domainand SH3 of FAK in its unphosphorylated form. The present invention forthe first time shows that AF can actively regulate the interaction ofCAP and FAK with NKCC1, giving rise to the occurrence of uncoupledphosphorylated FAK. By uncoupling FAK from the NKCC1 complex, the ionchannel is effectively shut down. The present inventors for the firsttime demonstrate strong evidence that AF can regulate the binding of CAPto flotillin 1 and 2, and to the SHP-2/PTPD1 domain of FAK and canpotentially interfere with the oligomerisation of flotillin 1 and 2 andits binding to the lipid raft in the cell membrane. AF is further by thepresent inventors for the first time shown to actively interfere withprotein phosphatases.

Through the above described biological action, AF can effectivelymonitor and/or normalize abnormal function of transmembrane proteins,such as the NKCC1 ion channel in the cell membrane.

In a healthy cell, the NKCC1/FAK/CAP complex shows a natural equilibriumbetween phosphorylated and unphosphorylated state, thus severalintracellular and extracellular effectors, such as receptors and osmoticpressure, can come to play and contribute to the finely tuned control ofthe state of the ion channel.

In pathological and/or perturbed cells, the finely tuned control of thestate of the ion channel NKCC1/FAK/CAP complex is disturbed and the ionchannel is constantly activated. Thus, the intracellular pressure ofe.g. transformed cells is often higher than that of healthy cells. Inpathological cells, FAK is constantly dephosphorylated and bound to theNKCC1 complex. Due to its unique potential to monitor FAK in the NKCC1complex, whereby FAK becomes phosphorylated, AF can effectivelynormalize the intracellular pressure in the pathological cell, and thuspotentially allow improved cellular uptake of a pharmaceuticalsubstance, such as drugs used in e.g. cancer therapy. Consequently,normalizing the intracellular pressure in the pathological cell can inturn also contribute to normalizing the interstitial pressure in apathological tissue. It ought to be stressed that abnormal intracellularlevels of ions results in occurrent transfer of water through e.g.aquaporins (water channels).

A well known drawback in the pharmacological treatment of a broadvariety of disease is the necessity to increase the dosage above optimumof the pharmaceutical substance administered to the patient due to thelack of an effective uptake of it by the pathological cells. The presentinvention makes it possible to utilize a proven non-toxic,biodegradable, endogenous small substance to normalize the intracellularpressure in the pathological cell, and thus potentially to allowimproved cellular uptake of any pharmaceutical substance, which in turnminimizes the risk for severe side effects due to administering anover-dose of said active further component.

Furthermore, the antisecretory factor (AF) has been proven to have noadversary effect on healthy normal cells, but only to exert itsnormalizing effect on pathological cells. On the contrary, theadministration of antisecretory factor (AF) protein, a homologue,derivative, and/or fragment thereof, having antisecretory and/orequivalent functional and/or analogue activity, or a pharmaceuticallyactive salt thereof can instead potentially even assist healthy cells inthe close vicinity of the pathological and/or perturbed cells to acquirean optimized equilibrium between phosphorylated and unphosphorylatedstates of NKCC1/FAK/CAP complex.

What is more, it is known since many years that a specific dietincluding a certain amount of malted cereals will induce the body's ownendogenous production of and/or activation of antisecretory factors inthe patient's blood. Thus, it is potentially possible to induce asimilar normalization of the intracellular pressure in the pathologicalcell, and/or the interstitial pressure in a pathological tissue, bysimply feeding the patients, which are to be treated with a certainsubstance or to be subjected to a certain treatment, with such foodstuffas described in e.g. WO 1998/21978 or in WO 05/030246 before, duringand/or after the treatment. Thus effectively and cheap improve thecellular uptake of a pharmaceutical substance, such as an anticancerdrug, radiation therapy, an antibiotic substance, an antiviral substanceor a drug targeting posttraumatic injury, neurodegeneration, a parasite,or an inflammatory condition.

The present invention further of course also relates to the use of theabove described surprising insight that antisecretory factor (AF) canintervene in the biological activation of transmembrane proteins, suchas ion channels, in particular NKCC1 through FAK in a broad variety ofmethods for improved drug design, for screening for and/or evaluatingpotential AF inhibitory and/or enhancing substances, and for evaluatingefficacy and/or verifying functional activity of new or knownantisecretory factor (AF) proteins, peptides, derivatives, homologues,and/or fragments thereof, having equivalent functional activity, and/ora pharmaceutically active salt thereof.

For example, phosphorylated FAK is easily distinguishable fromunphosphorylated FAK by specific and commercially available antibodies.As elegantly demonstrate by the present inventors, untreated MATB cells(AtCC N.: CRL-1666, designation 13762 MATB 111) in culture show clearlabeling for phosphorylated FAK. When they were exposed to a hypertonicsolution, which activated the cellular NKCC1 channel, the level ofphosphorylated FAK was markedly reduced, i.e. NKCC1 was activated andthe cells were swelling. In starch contrast hereto, treatment with AFnot only restored, but clearly induced a markedly higher level ofphosphorylated FAK in the cultured cells, i.e. NKCC1 was turned off andthe cells size became normalized.

Standard methods for improved drug design, for screening for and/orevaluating potential AF inhibitory and/or enhancing substances, and forevaluating efficacy and/or verifying functional activity of new or knownantisecretory factor (AF) proteins, peptides, derivatives, homologues,and/or fragments thereof, having equivalent functional activity, and/ora pharmaceutically active salt thereof are well known in the art, someof which are further described in the detailed section of thisapplication.

In another aspect, the present invention also relates to the use of apharmaceutical composition comprising an antisecretory factor (AF)protein, a homologue, derivative, and/or fragment thereof, havingequivalent functional activity, or a pharmaceutically active saltthereof, for the manufacture of a pharmaceutical composition for thetreatment and/or prevention of various medical conditions selected fromthe group consisting of medical conditions related to cancer, tumors ortumor related conditions, infections, inflammations, posttraumatic braininjury, neurodegeneration, and parasites.

In yet another aspect, the present invention relates to the use of thenewfound insight of AF's biological cellular function for the design ofa new and reliable diagnostic and/or prognostic tool for monitoringand/or verifying and/or enhancing the therapeutic control of a malignanttumor in a subject suffering from a neoplastic disease, such as cancer.

In a preferred embodiment, said antisecretory factor (AF) protein, to beused according to the present invention, consists of a sequenceaccording to the following formula

X1-V-C-X2-X3-K-X4-R-X5,

wherein X1 is I, amino acids 1-35 of SEQ ID NO 6, or is absent, X2 is H,R or K, X3 is S or L, X4 is T or A, X5 is amino acids 43-46, 43-51,43-80 or 43-163 of SEQ ID NO 6, or is absent, or a modification thereofnot altering the function of the polypeptide or peptide.

The invention is also related to various administration doses and routessuitable for the intended purpose of treatment as well as the patient'sage, gender, condition etc.

FIGURE LEGENDS

FIG. 1: Cytometric assay of free floating MATBIII tumor subjected tostress and AD treatment.

The FACS analyses, 10.000 cells, demonstrated a median FSC-height of 620in vial no. 1 (control), 450 in vial no. 2 (hypertonic NaCl 5 min), 514in vial no. 3 (Hypertonic NaCl+AF-16, 60 min) and 576 in vial no. 4(hypertonic NaCl+PBS, 60 min).

FIG. 2: Immunohistochemistry performed by means of antibodies tophosphorylated antibodies to FAK demonstrated an intense red staining inthe control cells (vial no 1), while the cells of vial no 4(PBS-treated) had a significantly lower intensity. A staining intensitysimilar to that in the control cells were demonstrated in the cellstreated with AF-16 (vial 3). A. Before treatment. B. Treatment withhypertonic NaCl followed by PBS. C. Treatment with hypertonic NaClfollowed by AF 16.

The cells are processed to demonstrate phosphorylated FAK (pFAK), redfluorescence. Nuclei are stained blue (DAPI).

Strong staining=high level of pFAK, i.e. the NKCC1 channel is closed.

Weak staining=low level of pFAK, i.e. the NKCC1 channel is open.

FIG. 3: GMK was cultured in on a solid surface in a series ofexperiments aimed to elucidate the presence and distribution of actinfilaments immunohistochemically with the aid of phalloidin-FITC.

A=Untreated, normal GMK cells cultured adherent to a surface (control)and processed for demonstration of actin filaments with aphalloidin-FITC conjugate. The nucleus is stained blue.

B=Treatment with cytochalasin B disintegrates the actin network and onlya few clusters remain in the cytoplasm

C=Concomitant treatment with cytochalasin B and AF-16 results in partialrestoring of the actin network. Thus AF-16 in parts normalize the cellsactin cytoskeleton

FIG. 4:

A=Vial 1 control

B=Vial 4 hypertonic NaCl+vehicle, 60 min

C=Vial 3 hypertonic NaCl+AF-16, 60 min

FIG. 5: Figure-Cryostat sections of MAT B III tumors cells from ratspre-treated for 60 min with either the vehicle (upper row, A-B) or AF-16(lower row, C-D), prior to the intravenous injection of doxorubicin. Therats were killed 15 min after injection of doxyrubicin. Binding ofdoxorubicin to DNA results in a red fluorescence in the nuclei of cellsexposed to the drug.

Only scattered nuclei are stained in tumors from rats pre-treated withthe vehicle (A and B). In tumor cells from rats pre-treated with AF-16most nuclei are stained. This shows that pre-treatment with AF-16efficiently increases the penetration of the cytostatic drug into thetumor cells' nuclei.

Bars=100 μm

DEFINITIONS AND ABBREVIATIONS Abbreviations

IFP: interstitial fluid pressure;

PBS: phosphate buffered saline;

AF: antisecretory factor,

AF-16: a peptide composed of the amino acids VCHSKTRSNPENNVGL; octapeptide IVCHSKTR; septa peptide VCHSKTR; hexa peptide CHSKTR; pentapeptide HSKTR.

FSC: Forward Scatter Count

SCS: Side Scatter Counts

SPC: Specially Processed Cereals

Definitions

Proteins are biological macromolecules constituted by amino acidresidues linked together by peptide bonds. Proteins, as linear polymersof amino acids, are also called polypeptides. Typically, proteins have50-800 amino acid residues and hence have molecular weights in the rangeof from about 6,000 to about several hundred thousand Dalton or more.Small proteins are called peptides, polypeptides, or oligopeptides. Theterms “protein”, “polypeptide”, “oligopeptide” and “peptide” may be usedinterchangeably in the present context.

A “pharmaceutical composition”, in the present context, refers to acomposition comprising a therapeutically active amount of anantisecretory factor (AF) protein, optionally in combination with apharmaceutically active excipient, such as a carrier or a vehicle. Saidpharmaceutical composition is formulated for the appropriate route ofadministration, which may vary depending on the condition of thepatient, as well as on other factors, such as age or preferred choice. Apharmaceutical composition comprising an antisecretory factor (AF)protein serves as a drug delivery system. The pharmaceutical compositionupon administration presents the active substance to the body of a humanor an animal. Said pharmaceutical composition may be in the form of e.g.tablets, pills, lozenges, capsules, stool pills, gels, solutions, etc,but is not limited thereto.

The term “pharmaceutically active salt”, refers to a salt of anantisecretory factor (AF) protein, which may be any salt derived therefrom, based on so-called Hofmeiser's series. Other examples ofpharmaceutically active salts comprise triflouroacetate, acetate andlysine chloride, the invention is not limited thereto.

The term “antisecretory” refers in the present context to inhibiting ordecreasing secretion, especially intestinal secretions. Hence, the term“antisecretory factor (AF) protein” refers to a protein capable ofinhibiting or decreasing secretion in a body.

In the present context, “equivalent functional and/or analogue activity”relates to the biological effect of the antisecretory factor (AF)protein, peptide, or polypeptide, or a homologue, derivative or fragmentthereof, i.e. its capacity for optimizing delivery and cellular uptakeof a pharmaceutical substance and/or formulation. Standardized examplesfor testing and/or measuring such a capacity are well known in the fieldof the art. Examples are given in the experimental section of thisapplication, such as in examples 1-6.

In the present context, an “antisecretory factor (AF) protein”, or ahomologue, derivative or fragment thereof, may be used interchangeablywith the term “antisecretory factors” or “antisecretory factor proteins”as defined in WO 97/08202, and refers to an antisecretory factor (AF)protein or a peptide or a homologue, derivative and/or fragment thereofhaving antisecretory and/or equivalent functional and/or analogueactivity, or to a modification thereof not altering the function of thepolypeptide. Hence, it is to be understood that an “antisecretoryfactor”, “antisecretory factor protein”, “antisecretory peptide”,“antisecretory fragment”, or an “antisecretory factor (AF) protein” inthe present context, also can refer to a derivative, homologue orfragment thereof. These terms may all be used interchangeably in thecontext of the present invention. Furthermore, in the present context,the term “antisecretory factor” may be abbreviated “AF”. Antisecretoryfactor (AF) protein in the present context also refers to a protein withantisecretory properties as previously defined in WO97/08202 and WO00/38535. Antisecretory factors have also been disclosed e.g. in WO05/030246. Also intended by the term antisecretory factor is egg yolkenriched in antisecretory factors as disclosed in SE 900028-2 and WO00/38535 as further described below.

A “medical food”, in the present context, refers to a food or a food forspecial dietary use, which has been prepared with a composition with anantisecretory factor (AF) protein or alternatively has the capability toinduce synthesis or activation of endogenous AF. Said food may be anysuitable food, in fluid or solid form, such as a liquid or a powder, orany other suitable foodstuff. Examples of such matter may be found in WO0038535 or WO 91/09536. Said constituent may as well induce the uptake,formation and release of an antisecretory factor (AF) protein.

A “nebulizer”, in the present context, refers to a medical device thatdelivers liquid medication in the form of a mist to the airways.“Nebulizer” compressors force air through tubing into a medicine cupfilled with liquid medicine. The force of the air breaks the liquid intotiny mist-like particles that can be inhaled deeply into the airways.

The term “aerosol” in the present context refers to a gaseous suspensionof fine solid or liquid particles.

In the present context, the term “cytostatica” is used, as well as“cytostatic drugs”, “Cytostatic agents” or “cytostatic compounds”, theterms are interchangeable and relate to drugs which are used in cancertherapy and are typically administerd to patients undergoingchemotherapy. Cytostatic agents are substances which check the growth ofpathological cells, but also of normal cells. Such substances aretherefore used for the chemotherapeutical treatment of tumors, but alsofor post-operational treatment after removal of a tumor. Cytostaticagents can come in liquid, powder or granular form, optionally alsodeep-frozen. The person skilled in the art will adjust the choice anddosage of cytostatica from a plethora of cytostatica commerciallyavailable.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a new approach to monitor cellfunctions related to systems in a cell controlling the cell's dimensionsand its internal milieu. An antisecretory factor (AF) protein, peptide,derivative, homologue, and/or fragment thereof, having equivalentfunctional activity, or a modification thereof not altering the functionof the polypeptide, and/or a pharmaceutically active salt thereof, isherein used for optimizing delivery and cellular uptake of apharmaceutical substance and/or formulation. Said pharmaceuticalsubstance and/or formulation is in the present context selected from thegroup consisting of anticancer drug, anti tumor drug, radiation therapy,antibiotic substance and antiviral substance, a drug targetingposttraumatic injury, a drug targeting neurodegeneration, a drugtargeting parasites, and a drug against inflammatory conditions.

In general, the present invention relates to the surprising insight thatAF can be used to correct the structure and function of cells that haveimpaired dimensions and interior cell milieu. For example, antisecretoryfactor (AF) has prior been shown to be able to intervene with thecytoskeleton and its associations to lipid rafts and caveolae, filamin,galectin and flotillin complexes and is now further described to beeffective in monitoring the activity of ion pumps such as NKCC1 byinteracting with CAP/ponsin (c-Cbl associated protein) and FAK (focaladhesion kinase). Without wishing to be limited to a single scientifichypothesis, it is herein envisioned that AF exerts at least part of itsregulating effect on NKCC1 through its previously documented influenceon the cellular levels of cAMP, which in turn effects the equilibriumbetween phosphorylated and dephosphorylated states of a vast number offunctional proteins, such as on CAP/ponsin (c-CBL associated protein)and/or FAK (focal adhesion kinase). The functionality of these proteinsis consequently influenced by the levels of AF. E.g. AF's effect on theequilibrium between phosphorylated and dephosphorylated states of FAKhas been demonstrated by the present inventors, and is documented in theexperimental section. What is more, the present inventors have furtherbeen able to demonstrate the association of AF to a phosphatase.

As shown in the experimental section, AF-16 could be demonstrated tocounteract deviations from the normal status for a cell, which giveshigh priority to turn normalized. The inventors thus unexpectedlydemonstrated the importance of the ability of AF-16 to turn a perturbedcell normalized, as revealed for a range of cell systems tissues andorgans.

AF effectively regulates and/or normalizes abnormal activity of said ionchannel in perturbed and pathological cells, thereby effectivelynormalizing the dimensions and the intracellular pressure in the cell,which in turn also can lead to normalizing the interstitial pressure ina perturbed tissue, and thus potentially allowing an improved cellularuptake of a pharmaceutical substance, such as drugs used in e.g. cancer,inflammation and trauma therapy.

The present invention further of course also relates to the use of theabove described surprising insight that antisecretory factor (AF) canintervene in the biological activation of cell systems regulatingdisturbed cell dimensions and internal cellular milieu through theregulation of NKCC1, by interacting with CAP/ponsin (c-Cbl associatedprotein) and FAK (focal adhesion kinase), in a broad variety of methodsfor improved drug design, for screening for and/or evaluating potentialAF inhibitory and/or enhancing substances, and for evaluating efficacyand/or verifying functional activity of new or known antisecretoryfactor (AF) proteins, peptides, derivatives, homologues, and/orfragments thereof, having equivalent functional activity, and/or apharmaceutically active salt thereof.

The present invention relates to the use of a pharmaceutical compositioncomprising an antisecretory factor (AF) protein, peptide, derivative,homologue, and/or fragment thereof, having equivalent functionalactivity, or a modification thereof not altering the function of thepolypeptide, and/or a pharmaceutically active salt thereof, for themanufacture of a pharmaceutical composition for optimizing deliveryand/or cellular uptake of a second or further pharmaceutical substanceand/or formulation. Typically, said second or further pharmaceuticalsubstance and/or formulation comprises an anticancer drug, radiationtherapy, antimicrobial substance, antibiotic substance, antiviralsubstance and/or a drug targeting posttraumatic injury,neurodegeneration, a parasite, and/or an inflammatory condition.

In yet another aspect, the present invention relates to a pharmaceuticalcomposition comprising an antisecretory factor (AF) protein, peptide,derivative, homologue, and/or fragment thereof, having equivalentfunctional activity, or a modification thereof not altering the functionof the polypeptide, and/or a pharmaceutically active salt thereof incombination with a second or further pharmaceutical substance and/orformulation, wherein said second or further pharmaceutical substanceand/or formulation is selected from the group consisting of ananticancer drug, radiation therapy, antibiotic substance, antiviralsubstance and a drug targeting posttraumatic injury, neurodegeneration,a parasite, or an inflammatory condition, as such, and to its use inmedicine, in particular to its use in the treatment of the variousmedical indications described in the present application.

Furthermore, said pharmaceutical composition can of course comprise twoor more antisecretory factor (AF) proteins, fragments or derivates, orcombinations thereof, as well as further comprising a pharmaceuticallyacceptable excipient.

In a presently preferred embodiment, antisecretory factor (AF) proteins,peptides, derivatives, homologues, and/or fragments thereof, havingequivalent functional activity, and/or a pharmaceutically active saltthereof, are shown to be able to overcome cellular barriers in malignantand/or pathological cells, and can thus be used for lowering a requireddrug dosage, alternatively for maximizing the dosage effect of saidpharmaceutical substance and/or formulation. In consequence, said abovedescribed AF can be used to minimize toxic or unwanted side effects ofsaid pharmaceutical substance and/or formulation.

The present invention relates to the use of a pharmaceutical compositioncomprising an antisecretory factor (AF) protein, a homologue,derivative, and/or fragment thereof, having antisecretory and/orequivalent functional and/or analogue activity, or a pharmaceuticallyactive salt thereof, for the manufacture of a pharmaceutical compositionfor optimizing delivery and/or cellular uptake of a second or furtherpharmaceutical substance and/or formulation. AF and the second orfurther pharmaceutical substance and/or formulation can be administeredtogether or in alternating succession. They can be co-formulated oradministered in separate formulations.

As documented in experiment 1, AF-16 transiently lowers the IFP during afew hours in tumor cells, thereafter IFP returns to the original highlevel in 24 h. Tentatively, this time limited effect of suppressed IFPis likely to be associated with improved tumor blood circulation andmetabolism, which might potentiate the efficacy of radio therapy. Thus,an increase of blood flow during radio therapy generates more freeradicals and these are most effective in eliminating the malignantcells.

A presently preferred embodiment of the present invention is thus theuse of a pharmaceutical composition comprising an antisecretory factor(AF) protein, a homologue, derivative, peptide and/or fragment thereof,according to the present invention, for the manufacture of apharmaceutical composition for optimizing radiation therapy.

What is more, based on the transient and reversible nature of thelowering of the cellular IFP, the clinician can easily envision anadministration routine wherein AF is administered in optimally timedintervals that are so adjusted that the IFP in the target cells arelowered just in time for the administration of the second or furtherpharmaceutical substance and/or formulation.

Thus, another, equally preferred embodiment relates to a method or to anadministration dosage regimen for optimized delivery and/or cellularuptake of a second or further pharmaceutical substance and/orformulation, wherein said second or further pharmaceutical substanceand/or formulation comprises an anticancer drug, radiation therapy,antibiotic substance, antiviral substance, and/or a drug targetingposttraumatic injury, neurodegeneration, a parasite, or an inflammatorycondition.

AF and the second or further pharmaceutical substance and/or formulationcan be administered together or in alternating succession. They can beco-formulated or administered in separate formulations.

In another aspect, the present invention relates to the use of apharmaceutical composition comprising an antisecretory factor (AF)protein, a homologue, derivative, and/or fragment thereof, havingequivalent functional activity, or a pharmaceutically active saltthereof, for the manufacture of a pharmaceutical composition foroptimizing delivery and/or cellular uptake of a second or furtherpharmaceutical substance and/or formulation for the treatment and/orprevention of various medical conditions selected from the groupconsisting of cancer, tumor or a tumor related condition, radiationtherapy, infection, posttraumatic injury, neurodegeneration, parasiticinfestation, and inflammatory conditions.

The invention is also related to various administration doses and routessuitable for the intended purpose of treatment as well as the patient'sage, gender, condition etc.

The pharmaceutical composition is herein formulated for intraocular,intranasal, oral, local, subcutaneous and/or systemic administration andcan e.g. be formulated for administration as a spray, aerosol, andinhaler or by a nebulizer. When formulated for administrationsystemically to the blood said composition is preferably formulated at adose of 0.1 μg to 10 mg per application and kg body weight and day, suchas at a dose of 0.1 μg to 1 mg per application and kg body weight andday, preferably again at 1-500 μg per application and kg body weight andday, such as at 1-50 μg, or 1-100 μg per application and kg body weightand day. Such an administration can be performed either as a single doseor as multiple daily applications.

The very wide range of effective dose regimes utilized indicates thatthe risks for side effects and unexpected complications are minimal.Thus, the present invention will enable the treatment of excessive loadson cells and tissues as wells as to treat a patient with a wide range ofdoses suiting the individual response and the severity of the illnessand/or the discomfort.

The pharmaceutical composition according to the present invention can inone context be administrated by application topically, locally in situ,orally, in the nose, subcutaneously and/or systemically via bloodvessels or via the respiratory tract.

The present invention further of course also relates to the use of theabove described surprising insight that antisecretory factor (AF) canintervene in the biological activation of NKCC1 and other transmembraneproteins through FAK in a broad variety of methods for improved drugdesign, for screening for and/or evaluating potential AF inhibitoryand/or enhancing substances, and for evaluating efficacy and/orverifying functional activity of new or known antisecretory factor (AF)proteins, peptides, derivatives, homologues, and/or fragments thereof,having equivalent functional activity, and/or a pharmaceutically activesalt thereof.

For example, phosphorylated FAK is easily distinguishable fromunphosphorylated FAK by specific and commercially available antibodies.As elegantly demonstrate by the present inventors, untreated MATB cells(established breast cancer cell line) in culture show clear labeling forphosphorylated FAK. When they were exposed to a hypertonic solution,which activated the cellular NKCC1 channel, the level of phosphorylatedFAK was markedly reduced, i.e. NKCC1 was activated and the cells wereswelling. In starch contrast hereto, treatment with AF not only restoredbut clearly induced a markedly higher level of phosphorylated FAK in thecultured cells, i.e. NKCC1 was turned off and the cells' sizenormalized.

The present invention thus in one presently preferred embodiment relatesto a method for improved drug design characterized by testing theresponse of cells or tissues, subject to treatment of a substance or apharmaceutical formulation referred to in the present application andestimating the influence of AF, antisecretory factor (AF) proteins,fragments or derivates, or combinations thereof on the cellular uptakeof said substance or formulation by e.g. measuring the amount ofphosphorylated FAK.

In another, equally preferred embodiment, the invention relates to amethod for screening for and/or evaluating potential AF inhibitoryand/or enhancing substances, characterized by selecting a chemical orbiological substance, exposing antisecretory factor (AF) proteins,peptides, derivatives, homologues, and/or fragments thereof, havingequivalent functional activity, and/or a pharmaceutically active saltthereof to said selected substance, and subsequently testing thepotential of said exposed AF to block the dephosphorylation of FAK atthe NKCC1 complex by measuring the quantitative occurrence ofphosphorylated FAK. In a presently specifically preferred embodiment,said testing method is performed as a high-throughput screening.

A further method is also related to herein, for evaluating the efficacyand/or verifying the functional activity of new or known antisecretoryfactor (AF) proteins, peptides, derivatives, homologues, and/orfragments thereof, having equivalent functional activity, and/or apharmaceutically active salt thereof, characterized by testing thepossibility of said new or known AF to block the dephosphorylation ofFAK at the NKCC1 complex by measuring and quantifying the occurrence ofphosphorylated FAK.

Any of the above described methods can typically alternatively beconducted in a cellular system or in a test organism. The methods arealso equally applicable in in vivo, in situ, and in silico systems.

Standard methods:

-   -   for improved drug design,    -   for screening for and/or evaluating potential AF inhibitory        and/or enhancing substances,    -   for evaluating efficacy and/or verifying functional activity of        new or known antisecretory factor (AF) proteins, peptides,        derivatives, homologues, and/or fragments thereof, having        equivalent functional activity, and/or a pharmaceutically active        salt thereof,        are well known in the art.

In yet another aspect, the present invention relates to the use of thenewfound insight of AF's biological cellular function for the design ofa new and reliable diagnostic and/or prognostic tool for monitoringand/or verifying and/or enhancing the therapeutic control of a malignanttumor in a subject suffering from cancer. In one embodiment of thepresent invention, antisecretory factor (AF) proteins, peptides,derivatives, homologues, and/or fragments thereof, having equivalentfunctional activity, are employed as markers for invasion and/ormetastasis of various tumor types and/or cancer forms.

The direct regulatory effect of antisecretory factor (AF) proteins,peptides, derivatives, homologues, and/or fragments thereof, havingequivalent functional activity, and/or a pharmaceutically active saltthereof on the phosphorylation state of FAK in particular, renders thesaid AF itself to be a potential drug candidate for cancer and/or tumortreatment.

The antisecretory factor is a class of proteins that occur naturally inthe body. The human antisecretory factor protein is a 41 kDa protein,comprising 382-288 amino acids when isolated from the pituitary gland.The active site with regard to the beneficial effect on normalization ofNKCC1 according to the present invention can be localized to the proteinin a region close to the N-terminal of the protein, in particularlocalized to amino acids 1-163 of SEQ ID NO 6, more specifically toamino acid positions 35-50 on the antisecretory factor (AF) proteinsequence. The biological effect being exerted by any peptide orpolypeptide comprising at least 4-6 amino acids of said consensussequence, or a modification thereof not altering the function of thepolypeptide and/or peptide.

The present inventors have shown that the antisecretory factor is tosome extent homologous with the protein S5a, and Rpn10, whichconstitutes a subunit of a constituent prevailing in all cells, the 26 Sproteasome, more specifically in the 19 S/PA 700 cap. In the presentinvention, antisecretory factor (AF) proteins are defined as a class ofhomologue proteins having the same functional properties. Theproteasomes have a multitude of functions related to the degradation ofsurplus proteins as well as short-lived unwanted, denatured, misfoldedand otherwise abnormal proteins. Further, the antisecretoryfactor/S5a/Rpn10 is involved in the distribution and transportation ofcell constituents, most evidently proteins. Antisecretory factor is alsohighly similar to angiocidin, another protein isoform known to bind tothrombospondin-1 and associated with cancer progression.

Homologues, derivatives and fragments of antisecretory factor (AF)proteins and/or peptides according to the present invention all haveanalogous biological activity of being able to be used for themanufacture of a medicament for the food for optimizing delivery and/orcellular uptake of a further pharmaceutical substance and/orformulation, as well as in a method for treating conditions such astumors and tumor-related conditions, infections, inflammations and/orconditions caused by parasites. Homologues, derivatives and fragments,in the present context, comprise at least 4 amino acids of a naturallyoccurring antisecretory factor (AF) protein, which may be furthermodified by changing one or more amino acids in order to optimize theantisecretory factor's biological activity for optimizing deliveryand/or cellular uptake of a further pharmaceutical substance and/orformulation related to the present invention, without altering theessential function of the polypeptide and/or peptide.

A fragment of an antisecretory factor (AF) protein will generallycomprise the peptide/amino acid sequence or a fragment thereof in apreparation in which more than 90%, e.g. 95%, 96%, 97%, 98% or 99% ofthe protein in the preparation is a protein, peptide and/or fragmentsthereof of the invention.

Furthermore, any amino acid sequence being at least 70% identical, suchas being at least 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical with the amino acid sequenceof a antisecretory factor (AF) protein, peptide, homologue, derivativeand/or fragment according to the invention, is also considered to beinside the scope of the present invention. In the present context theterms homologous and identity are used interchangeably, i.e. an aminoacid sequence having a specified degree of identity with another aminoacid sequence has the same degree of homology to a specified amino acidsequence.

By a derivative is in the present context intended a protein havingequivalent activity and/or a functional equivalent activity to anantisecretory factor as defined herein, being derived from anothersubstance either directly or by modification or partial substitution,wherein one or more amino acids have been substituted by another aminoacid, which amino acid can be a modified or an unnatural amino acid. Forexample, the antisecretory factor derivatives according to the inventionmay comprise an N terminal and/or a C terminal protecting group. Oneexample of an N terminal protecting group includes acetyl. One exampleof a C terminal protecting group includes amide.

By proteins, homologues, derivatives, peptides and/or fragment thereofhaving an amino acid sequence at least, for example 95% identical to areference amino acid sequence, is intended that the amino acid sequenceof e.g. the peptide is identical to the reference sequence, except thatthe amino acid sequence may include up to 5 point mutations per each 100amino acids of the reference amino acid sequence. In other words, toobtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the aminoacids in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acids in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among amino acids in the reference sequence or in one ormore contiguous groups within the reference sequence.

In the present invention, a local algorithm program is best suited todetermine identity. Local algorithm programs, (such as Smith Waterman)compare a subsequence in one sequence with a subsequence in a secondsequence, and find the combination of sub-sequences and the alignment ofthose sub-sequences, which yields the highest overall similarity score.Internal gaps, if allowed, are penalized. Local algorithms work well forcomparing two multidomain proteins, which have a single domain, or justa binding site in common.

Methods to determine identity and similarity are codified in publiclyavailable programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package (Devereux, J et al (1994)) BLASTP,BLASTN, and FASTA (Altschul, S. F. et al (1990)). The BLASTX program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,S. F. et al, Altschul, S. F. et al (1990)). Each sequence analysisprogram has a default scoring matrix and default gap penalties. Ingeneral, a molecular biologist would be expected to use the defaultsettings established by the software program used.

The antisecretory factor (AF) proteins or a peptide or a homologue,derivative and/or fragment thereof having equivalent activity as definedherein, can comprise 4 amino acids or more, such as 5-16 amino acids,such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20amino acids or more. In other preferred embodiments the antisecretoryfactor consists of 42, 43, 45, 46, 51, 80, 128, 129 or 163 amino acids.In preferred embodiments the antisecretory factor (AF) consists of 5, 6,7, 8 or 16 amino acids.

In another preferred embodiment, the antisecretory factor (AF) proteinsor a peptide or a homologue, derivative or fragment thereof havingequivalent activity according to the present invention consists of asequence according to the following formulae:

X1-V-C-X2-X3-K-X4-R-X5

wherein X1 is I, amino acids 1-35 of SEQ ID NO 6, or is absent, X2 is H,R or K, X3 is S or L, X4 is T or A, X5 is amino acids 43-46, 43-51,43-80 or 43-163 of SEQ ID NO 6, or is absent.

The antisecretory factor according to the present invention, can beproduced in vivo or in vitro, e.g. recombinantly, synthetically and/orchemically synthesized, and/or isolated from a naturally occurringsource of antisecretory factors, such as from pig pituitary glands orbird's eggs. After production, the antisecretory factors may be furtherprocessed, such as by chemical or enzymatic cleavage to smallerantisecretory active fragments or by modification of amino acids. It ispresently not possible to obtain antisecretory factor (AF) protein inpure form by purification. It is however possible to produce abiologically active antisecretory factor protein recombinantly orsynthetically as previously disclosed in WO 97/08202 and WO 05/030246.WO 97/08202 also discloses the production of biologically activefragments of this protein of 7-80 amino acids.

The antisecretory factor according to the invention may further comprisean N terminal and/or a C terminal protecting group. One example of an Nterminal protecting group includes acetyl. One example of a C terminalprotecting group includes amide.

In a preferred embodiment of the present invention the antisecretoryfactor is a selected among SEQ ID NO 1-6, i.e. VCHSKTRSNPENNVGL (SEQ IDNO 1, in this context also called AF-16), IVCHSKTR (SEQ ID NO 2),VCHSKTR (SEQ ID NO 3), CHSKTR (SEQ ID NO 4), HSKTR (SEQ ID NO 5), or theamino acid sequence of an antisecretory factor (AF) protein according toSEQ ID NO 6 using the common one letter abbreviations for amino acids.SEQ ID NO 1, 2, and 3 have previously been disclosed in e.g. WO05/030246. As specified in the accompanying sequence listing, some ofthe amino acids in the above-specified sequences may be replaced byother amino acids. In the following in this paragraph, the position of aparticular amino acid in a particular amino acid sequence is calculatedfrom the left, denoting the most N-terminal amino acid as being inposition 1 in that particular sequence. Any amino acid substitution(s)as specified below may be performed independently of any other aminoacid substitution(s) in that sequence. In SEQ ID NO 1, the C in position2 may be replaced by S, H in position 3 may be replaced with R or K, Sin position 4 may be replaced with L, and/or T in position 6 may bereplaced with A. In SEQ ID NO 2, C in position 3 may be replaced by S, Hin position 4 may be replaced by R or K, S in position 5 may be replacedby L, and/or T in position 7 may be replaced by A. In SEQ ID NO 3, C inposition 2 may be replaced by S, H in position 3 may be replaced by R orK, S in position 4 may be replaced by L, and/or T in position 6 may bereplaced by A. In SEQ ID NO 4, C in position 1 may be replaced by S, Hin position 2 may be replaced by R or K, S in position 3 may be replacedby L, and/or T in position 5 may be replaced by A. In SEQ ID NO 5, H inposition 1 may be replaced by R or K, S in position 2 may be replaced byL, and/or T in position 4 may be replaced by A.

Also intended by the present invention is the combination of two or moreof any of the fragments according to SEQ ID NO 1-6.

In one embodiment of the present invention, the pharmaceuticalcomposition according to the invention further comprises apharmaceutically acceptable excipient. The choice of pharmaceuticallyacceptable excipient and their optimum concentration for use accordingto the present invention can readily be determined by the skilled personby experimentation. Pharmaceutically acceptable excipents for useaccording to the present invention include solvents, buffering agents,preservatives, chelating agents, antioxidants, and stabilizers,emulsifying agents, suspending agents and/or diluents. Thepharmaceutical compositions of the invention may be formulated accordingto conventional pharmaceutical practice, e.g. according to “Remington:The science and practice of pharmacy”, 21st edition, ISBN 0-7817-4673-6or “Encyclopedia of pharmaceutical technology”, 2nd edition, ed.Swarbrick J., ISBN: 0-8247-2152-7. A pharmaceutically acceptableexcipient is a substance that is substantially harmless to theindividual to which the composition is to be administered. Such anexcipient normally fulfils the requirements given by the national healthauthorities. Official pharmacopoeias such as e.g. the BritishPharmacopoeia, the United States of America Pharmacopoeia and TheEuropean Pharmacopoeia set standards for pharmaceutically acceptableexcipients.

The following is a review of relevant compositions for optional use in apharmaceutical composition according to the invention. The review isbased on the particular route of administration. However, it isappreciated that in those cases where a pharmaceutically acceptableexcipient may be employed in different dosage forms or compositions, theapplication of a particular pharmaceutically acceptable excipient is notlimited to a particular dosage form or of a particular function of theexcipient. It should be emphasized that the invention is not limited tothe use of the compositions mentioned in the following.

Parenteral Compositions:

For systemic application, the compositions according to the inventionmay contain conventional non-toxic pharmaceutically acceptable carriersand excipients, including micro spheres and liposomes.

The compositions for use according to the invention may include allkinds of solid, semi-solid and fluid compositions.

The pharmaceutically acceptable excipients may include solvents,buffering agents, preservatives, chelating agents, antioxidants, andstabilizers, emulsifying agents, suspending agents and/or diluents.Examples of the different agents are given bellow.

Example of Various Agents:

Examples of solvents include but are not limited to water, alcohols,blood, plasma, spinal fluid, ascites fluid and lymph fluid.

Examples of buffering agents include but are not limited to citric acid,acetic acid, tartaric acid, lactic acid, hydrogen phosphoric acid,bicarbonates, phosphates, diethylamide, etc.

Examples of chelating agents include but are not limited to EDTA andcitric acid.

Examples of antioxidants include but are not limited to butylatedhydroxyl anisole (BHA), ascorbic acid and derivatives thereof,tocopherol and derivatives thereof, cysteine, and mixtures thereof.

Examples of diluents and disintegrating agents include but are notlimited to lactose, saccharose, emdex, calcium phosphates, calciumcarbonate, calcium sulphate, mannitol, starches and microcrystallinecellulose.

Examples of binding agents include but are not limited to saccharose,sorbitol, gum acacia, sodium alginate, gelatine, chitosan, starches,cellulose, carboxymethylcellulose, methylcellulose,hydroxypropylcellulose, polyvinylpyrrolidone and polyetyleneglycol.

The pharmaceutical composition according to the invention is can in onecontext be administrated locally or via intravenous peripheral infusionor via intramuscular or subcutaneous injection into the patient or viabuccal, pulmonary, nasal, cutaneous or oral routes. Furthermore, it isalso possible to administer the pharmaceutical composition through asurgically inserted shunt into a cerebral ventricle of the patient.

In one embodiment, the pharmaceutical composition used according to thepresent invention is formulated for intraocular, local, intranasal,oral, subcutaneous and/or systemic administration. In a preferredembodiment, the composition of the invention is administrated byapplication as a suspension or, even more preferably, a powder forinhalation with a spray, aerosol, inhaler or nebulizer nasally and/or tothe respiratory tract.

The administration of a powder comprising antisecretory factors has theadditional advantages in terms of stability and dosage. A pharmaceuticalcomposition according to the invention can also be topically applied,intraocularly, intranasally, orally, subcutaneously and/or systemicallyadministered via blood vessels. In a preferred embodiment, thepharmaceutical composition is formulated for intravenous, intramuscular,local, oral or nasal administration. Typically, when used for topicalapplication to the eye, the applied concentration in the composition ofthe invention is from 1 μg to 1 mg per application, preferably 50-250μg, either as a single dose per day or repeated several times per day(multiple doses), but is not limited thereto.

Systemically administrated to the blood, the dose is within the range of0.1 μg to 10 mg per application and kg body weight, such as 0.1 μg to 1mg per application and kg body weight, preferably 1-500 μg/kg bodyweight, preferably again 1-100 μg/kg body weight either as a single doseper day or repeated several times per day. When egg yolk enriched inantisecretory factors is used according to the present invention, thisformulation is preferably administered orally.

Accordingly, the present invention relates to the use of anantisecretory factor (AF) protein or a derivative, homologue, and/orfragment thereof, having equivalent activity, and/or a pharmaceuticallyactive salt thereof, for optimizing delivery and/or cellular uptake of afurther pharmaceutical substance and/or formulation. In one embodiment,said antisecretory factor (AF) protein consists of a sequence accordingto the following formula

X1-V-C-X2-X3-K-X4-R-X5

wherein X1 is I, amino acids 1-35 of SEQ ID NO 6, or is absent, X2 is H,R or K, X3 is S or L, X4 is T or A, X5 is amino acids 43-46, 43-51,43-80 or 43-163 of SEQ ID NO 6, or is absent. In another embodiment, theinvention relates to the use of an antisecretory factor (AF) proteinwhich comprises an amino acid sequence as shown in SEQ ID NO: 1. Inanother embodiment, the invention relates to the use of an antisecretoryfactor (AF) protein which comprises an amino acid sequence as shown inSEQ ID NO: 2. In yet another embodiment, the invention relates to theuse of an antisecretory factor (AF) protein which comprises an aminoacid sequence as shown in SEQ ID NO: 3. In yet another embodiment, theinvention relates to the use of an antisecretory factor (AF) proteinwhich comprises an amino acid sequence as shown in SEQ ID NO: 4. In ayet further embodiment, the invention pertains to the use or anantisecretory factor (AF) protein which comprises an amino acid sequenceas shown in SEQ ID NO: 5.

Furthermore, in yet another embodiment, the invention pertains to theuse of an antisecretory factor (AF) protein which is a protein with anamino acid sequence as shown in SEQ ID NO 6, or a homologue, derivativeand/or fragment thereof comprising amino acids 38-42 of SEQ ID NO 6.

In yet another embodiment, the invention relates to the use of apharmaceutical composition as disclosed herein, which comprises two ormore antisecretory factor (AF) proteins selected from the proteins asdisclosed in SEQ ID NO: 1-6, and SEQ ID NO 6 or a homologue, derivativeand/or fragment thereof comprising amino acids 38-42 of SEQ ID NO 6, ora sequence as disclosed by the general formulae described herein. Saidsequences are all equally preferred to be used in the present invention.

In one embodiment of the invention, said pharmaceutical compositionfurther comprises a pharmaceutically acceptable excipient. Such anexcipient may be any preferable excipient chosen to be appropriate forthe specific purpose. Examples of excipients are disclosed herein.

In another embodiment of the invention, said pharmaceutical compositionis formulated for intraocular, intranasal, oral, local, subcutaneousand/or systemic administration. The chosen route of administration willvary depending on the condition of the patient to be treated and thepatient's age and gender etc.

In another embodiment, the pharmaceutical composition is formulated foradministration as a spray, aerosol or by a nebulizer or an inhaler. Inyet another embodiment, the invention relates to a pharmaceuticalcomposition and/or a medical food which is formulated for administrationsystemically to the blood at a dose of 0.1 μg to 10 mg per applicationand kg body weight and day, such as 0.1 μg to 1 mg per application andkg body weight and day, preferably 1-500 μg per application and kg bodyweight and day, preferably again 1-50 μg per application and kg bodyweight and day. In another embodiment, said dose is 1-1000 μg perapplication and kg body weight and day, such as 1-100 μg per applicationand kg body weight and day. The amount of the pharmaceutical compositionwhich is distributed to the patient in need thereof will of course varydepending on the patient to be treated, and will be decided by theskilled person, such as a medical practitioner, for each occasion. Saidadministration can be performed either as a single dose or as multipledaily applications.

EXPERIMENTAL SECTION Example 1 Disintegration of the Actin Filaments inIntestinal Epithelial Cells

The aim of the present experiment was to elucidate if disintegration ofthe actin filaments in intestinal epithelial cells (enterocytes)influenced the fluid secretion in the small intestine in adult rats.

Materials and Methods

Cholera toxin (Sigma), 3 μg, was infused in a 15 cm long ligated loopformed from the jejunum. Such a treatment resulted in 5 h inaccumulation of fluid, which was measured to amount to 340 mg/cmintestinal length. The actin network, normally distinctly and strictlyorganized in the epithelial cells, was thoroughly disorganized. Themicrovilli on the luminal surface of the intestinal epithelial cellswere short, clumpy and irregular in their outline while their core ofmicrovilli almost completely lacked.

Results

If the intestinal loop was challenged with cholera toxin and then hadAF-16, 100 μg, infused systemically within 10 minutes, the expectedhypersecretion was abolished. The weight of the loop was roughly equalto that of the tissue after challenge with buffered balanced saline, andno fluid accumulation or inflammatory reactions could be recognized.Thus, AF-16 completely inhibited the expected fluid hypersecretion.

If on the other hand, the same amount of AF-16 was delivered 30 minutesafter the cholera toxin challenge, the fluid accumulated in the loop wasin the order of 320-350 mg/cm loop length. The actin network in theenterocytes, as well as in the microvilli was extensively disorganized.We conclude that there is a time window related to the hypersecretionpreventing effects of AF-16 after cholera toxin challenge. These resultunequivocally disclosed that AF-16 only was preventing intestinal fluidhypersecretion during the first 10 minutes to, at longest, 15 minutes,after its administration. Intestinal fluid hypersecretion is linked tothe prevalence of disorganized actin network and some swelling of theenterocytes.

Combined challenge with cholera toxin and methyl-β-cyclodextrin (MβCD)of a ligated small intestine loop in rats resulted in the accumulationof 480-500 mg fluid per cm length of loop, and a prominent inflammationof the tissue. The actin filaments were extensively disorganized. MβCDis known to deplete lipid rafts of cholesterol, resulting in dissipationand aggregation of lipid raft constituents as well as abnormal functionsof ion pumps and other transduction signaling receptors prevalent inthese structures. The actin cytoskeleton was disorganized.

Combined challenge with cholera toxin and methyl-β-cyclodextrin (MβCD)of a ligated small intestine loop in rats followed within 10 minutes ofadministration of 100 μg AF-16 resulted in the accumulation of less than100 mg fluid per cm length of loop, and no obvious inflammation of thetissue. The actin filaments were distinctly organized both in the apicalparts of the intestocytes and in the almost normally appearingmicrovilli. We conclude that disorganization of the actin network and ofthe lipid rafts results in extensive fluid hypersecretion, possible tocompletely prevent by the administration of a sufficient single dose ofAF-16, if administrated within 10-15 minutes.

Example 2 Effect of AF-16 on Tumor Cell Distribution of Doxorubicin

The effect of AF-16 on the vascular and cellular supply of a cytotoxic,low molecular weight drug in the tumor was investigated by usingdoxorubicin, which emits a distinct, red fluorescence and binds to DNA.The frequency of red labeled cell nuclei was used as a measure of theaccess of doxorubicin to and into individual tumor cells. Animals withMat B III tumors were pretreated by either an intranasal dose of 100 μgAF-16 (n=3) or the vehicle, PBS (n=3) 60 min prior to a single i.v.injection of doxorubicin (9 mg/kg b.w). The animals were sacrificedafter 15 min, the tumors were dissected out and snap frozen in liquidnitrogen. Cryostat microtome sections were prepared, attached to glassslides and fixed in 4% buffered formaldehyde. After staining nuclei withHoechst 33342 (Sigma), the sections were mounted with Vectashield™(Vector Laboratories Inc, USA) and examined in a Zeiss Axio10fluorescence microscope.

Materials and Methods

Animals and tumors. Female rats of the Fisher 344 and Sprague-Dawleystrains were purchased from B & K, Stockholm, Sweden. The animals werehoused with a 12 h light cycle, standardized humidity and temperature,and with access to pellets and water ad libitum.

MAT B III (ATCC; CRL-1666; #13762) syngenic tumors. These tumors wereestablished in female Fisher rats (160 gram b.w.) after a singlesubcutaneous injection between the shoulder blades of 106 MAT B IIIcells dissolved in 0.2 ml culture medium. One or more solid tumorsdeveloped in 10-12 days, and reached in 2-5 days a size of 10×8×5 mm,enabling pressure recordings.

Ethics. Permits to perform experiments were granted by the regionalAnimal Experiments Ethics Committee, and local and federal guidelines(EU 86/609/EEC) were followed.

Chemicals. The peptide AF-16 was synthesized and characterized by RossPedersen A/S, Copenhagen, Denmark. DMBA (9, 10 dimethyl-1,2-benzanthracene, Hoechst 33342) and doxorubicin were purchased fromSigma. The cell culture medium RPMI 1640, supplemented with L-glutamine,was purchased from Flow Lab.

Histopathology and immunohistochemistry. At the end of the experimentsthe tumors were dissected, isolated free and either rapidly frozen inliquid nitrogen or fixed in 4% buffered formaldehyde. The fixed tumorswere embedded in paraffin, sectioned and stained with hematoxylin andeosin.

Statistics: Results are given as means±SE. Paired design tests wereused, and p<0.05 was considered significant.

Results

Effect of AF-16 on the distribution of doxorubicin. Doxorubicin bound toDNA can be recognized by the bright, red fluorescence of the cellnuclei. In two out of three MAT B III tumors from rats pretreated withAF-16 the frequency of positive, red nuclei was higher than in thetumors from rats pretreated with the vehicle (FIG. 5). The blue dyeHoechst 33342 stain all cell nuclei, enabling at the microscopicinspection of tumors to select areas with equal density of cells. TheFIGS. 5A/B and C/D, respectively, have the same density of tumor cells,but the specimen from an animal pretreated with AF-16 display stronglyincreased occurrences of red nuclei. This experiment thus revealed thatAF-16 enables doxorubicin to reach close to every cells at sufficientconcentration. We further conclude that these results indicated thatAF-16 in addition facilitates the optimized delivery and/or cellularuptake of a low molecular weight drug into the individual cells of thetumor tissue.

Example 3

The aim of these experiments was to elucidate alterations of the cellsurface area, i.e. of the cell volumes, as related to the tonicity ofthe fluid medium in which the MATB cells were suspended. Tumor cells, asdo normal cells, give high priority to maintain their cell volumesexactly regulated. The utilized strain of MATB cells is known to largelyrely on the use of the fluid and ion transfer pump NKCC-1 for monitoringtheir dimensions and internal milieu.

Materials and Methods

The tumor cell line MATB cells (ATCC Nr: CRL-1666, designation 13762MATB 111), a well known and commonly used cell line, was cultured insuspension and used in cell flow experiments using a FACS equipment.Thereby, the close to spherical tumor cells could rapidly alter theirsurface area and cell volume as a response to challenges through theextracellular fluid.

The FACS apparatus measured the MATB cells surface to be 620 units inisotonic medium, lacking any serum or other external proteins andpeptide constituents that could interfere. The addition of 10% sodiumchloride (NaCl) to the suspension medium resulted in 5 minutes in ashrinkage of the MATB cells surface are, to just below 500 units. Thatactivated the NKCC-1 ion pump system as the cells sensed their dimensionreductions. The FAK and the CAP systems are connected to the flotillins,linked to the actin filaments in the MATB cells. Activation of thissystem results in phosphorylation of NKCC-1/FAK/CAP complex, and theMATB cells ingest water and ions to swell. The cells struggle intenselyand take all opportunities to regain the normal cell volume. Thereby,most evidently Na⁺ and water enable the cell to increase its cellsurface to 576 units, i.e. close to that measured at the beginning ofthe experiment. If, however, AF-16, 100 μg/mL, is added after 30minutes, the activity of the NKCC-1 pump in the lipid rafts is rapidlyreduced as NKCC-1 is dephosphorylated and thereby inactivated, asreflected by the cell surface of the MATB cells, which approaches 514units. These alterations are significant as based upon more than 1000cells per cycle.

Results

Immunohistochemical processing of MATB cells centrifuged to be depositedon glass slides confirmed the dynamics of the interaction between theCAP-FAK-NKCC-1 systems. The GMK cells had high prevalence ofphosphorylated FAK in isotonic solution prior to the challenge by theaddition of saline, turning the suspension medium hypertonic. Theexpression of phosphorylated FAK was almost completely abolished afterthe treatment with hypertonic solution, reflecting that the NKCC-1 ionpump with AF-16 restored the high expression of phosphorylated FAK,disclosing that the NKCC-1 pump system was shut down.

We conclude that the exposure of a suspension of MATB cells to ahypertonic solution activated the CAP-FAK system, which monitors theactivity of the NKCC-1 ion pump system. Thereby, we demonstrated thatAF-16 interferes with this system by controlling the activity of theNKCC-1 ion pump, by monitoring the CAP/ponsin and FAK system.

Example 5

The tumor cell line GMK was cultured in on a solid surface in a seriesof experiments aimed to elucidate the presence and distribution of actinfilaments immunohistochemically with phalloidin-FITC.

Materials and Methods

The GMK cells were harvested after culturing to sub confluence. The cellcultures were then, in triplicate, treated according to thespecifications in the following Table 1. After 30 min of treatment thecell cultures were fixed in buffered formalin and then processed forimmunohistochemical demonstration of the actin pattern, as visualizedwith labeled phalloidin. The latter is linked with high affinity toactin filaments but not to depolymerized or G-actin. The achievedresults were evaluated and photographed with the aid of a fluorescencemicroscope. (FIG. 3)

Results

We conclude that cytochalasin B disorganize the normal actin filamentpattern in cultured, adherent GMK cells and that treatment with AF-16 toa large extent restored the normal pattern. Thus AF-16 normalized aperturbed actin cytoskeleton.

TABLE 1 Bundles Lamellipodium of actin Submembranous with actin filamentDense Actin at Treatment actin rim filaments in cytoplasm bodiescytocentrum Comments Control ++ +++ +++ − + Actin at (sham Not thetreatment, visible position Hank's) of lipid rafts in plasma membrane100 μg AF- ++ +++ +++ − + No 16 per mL change 20 μM − − − ++ +++ Actincytochalasin pattern B during disorganized 30 min DMSO in ++ +++ +++ − +No Hank's change 100 μg AF- + − − ++ ++ Actin 16 per mL pattern 5 minprior disorganized to 20 μM cytochalasin B added 100 μg AF- ++ + + + ++Actin at 16 per mL plasma and 20 μM membrane cytochalasin B addedconcomitantly 20 μM +++ +++ +/++ −/+ + Actin at cytochalasin plasma B 5min membrane prior to the addition of AF-16

Example 6 Effects of AF-16 on the Cellular Volumetric Regulation ofMATB1 Cells In Vitro Materials and Methods Cell Preparation

MATB cells were cultivated according to standard procedures, and dilutedto 1×106 per ml in RPMI substituted with glutamine. The cells weredivided into 4 vials, designated no. 1, 2, 3 and 4. Vial 1 served as acontrol. To vial no. 2, 3 and 4 were added hypertonic NaCl (10 mg/ml inRPMI with glutamine), followed by addition of 50 mg of AF-16 (vial 3) orPBS (vial 4) after 30 min. Vial no 2 was subjected to FACS analyses 5min after addition of the hypertonic NaCl solution, while the cells invials 1, 3 and 4 were subjected to FACS analyses after a total of 60min. Cytospin preparations were made from all vials and used forimmunohistochemistry.

Results

The FACS analyses, of 10.000 cells, demonstrated a median FSC-height of620 in vial no. 1 (control), 450 in vial no. 2 (hypertonic NaCl 5 min),514 in vial no. 3 (hypertonic NaCl+AF-16, 60 min) and 576 in vial no. 4(hypertonic NaCl+PBS, 60 min) (FIG. 1). Immunohistochemistry performedby means of antibodies to phosphorylated FAK demonstrated an intense redstaining in the control cells (vial no 1), while the cells of vial no 4(PBS-treated) had a significantly lower intensity. A staining intensitysimilar to that in the control cells were demonstrated in the cellstreated with AF-16 (vial 3) (FIG. 2).

Interpretation of the Results.

Free floating MAT B III tumor cells were used in cytometry assays, andexposed to hypertonic stress, aimed to mimic a similar situation in thesolid tumor. The experimental setup was designed to elucidate if cellswelling, i.e. intracellular accumulation of fluid, contributed to thehigh IFP. Thus, the cells in an encapsulated tumor are considered to beinflated, maintaining this state of shape and size with a high priorityby pumping fluid and ions in order to counteract influences of pH andosmotic forces.

Thus, suspended MAT B III cells were exposed to hypertonic environment,induced by adding sodium chloride to the nutrient medium. Flow cytometrydisclosed a rapid decrease of the mean cell volume (FIG. 1). Within 10min, the volume regulating system of the MAT B III cells started toregain cell volume, approaching their original cell volume after 60 min.However, the addition at 30 min after the start of the hypertonicchallenge of AF-16 to the MAT BIII cells blocked the reactive increaseof the cell volume. Addition of AF-16 to the cell medium did notinfluence the volume of the free floating MAT BIII cells if added priorto the induction of hypertonic stress (not illustrated). The lack ofeffect when AF-16 was added before the reactive recovery had startedindicated that the fluid transfer system affected by AF-16 wastemporarily inactivated until the volume recovery started. Thisexperimental setup was repeated three times with the similar results.Taken together, these results demonstrated that AF-16 can affect thecell systems monitoring the dimensions and volume of tumor cells. Noinfluence of AF-16 on the cell volume has been documented in vitro whentested on different normal, adult cells.

The maintenance of cells size and shape is of key importance, not onlyfor normal cells but also for malignant ones. Cells respond rapidly whenthe environment is changed. Thus, the suspended MAT B III cells in thepresent experiment responded to a hypertonic environment by shrinkage.However, a counter reaction was rapidly started in order to restore thecells dimensions by increased uptake of fluid. However, the addition ofAF-16 blocked this active cell volume expansion in an hypertonic milieu.The decrease in IFP pressure documented after AF-16 treatment could bedue to an effect on the tumors cells, on the blood vessels or acombination of both. The flow cytometric results indicate that oneeffect could be a direct effect on the tumor cells reducing their volumeand thus facilitating perfusion through the tumors. Such an effect hasbeen demonstrated in tumors with pharmacologically induced elevatedfrequency of apoptosis causing a decrease of the tumor mass, whicheventually resulted in an improved blood perfusion (Jain 2008). High IFPin solid tumors is often correlated to hampered penetration and unevendistribution of cytotoxic drugs. These restrictions in the drugdistribution are frequently followed by a reduced anti tumor efficacy ofthe cytotoxic drugs.

It ought to be stressed that AF-16 transiently lowered the IFP during afew hours, thereafter IFP returned to the original high level in 24 h.Tentatively, this time limited effect of suppressed IFP is likely to beassociated with improved tumor blood circulation and metabolism, whichmight potentiate the efficacy of radio therapy. Thus, an increase ofblood flow during radio therapy generates more free radicals and theseare most effective in eliminating the malignant cells.

The flow cytometry results indicated that AF-16 directly or indirectlyaffects essential ion and water transport systems in the tumor cellsand/or in the stroma. A likely candidate could be the co-transporterNKCC-1, known to affect the volume of cells.

Addition of hypertonic NaCl induced an immediate shrinkage of the MATBcells due to altered osmotic pressure in the extracellular medium,leading to an activation of the NKCC1 pump, aiming at increasing the Na+and water concentration into the cell, counteracting the cell shrinkage.This activation is preceded by a dephosphorylation of FAK simultaneouslywith a phosphorylation of NKCC1 and other transmembrane proteins. Such adephosphorylation of FAK is inhibited by addition of AF-16. Anotherenzyme system, named CAP=ponsin, interacts with FAK as well as with theflotillin complex. Thus, AF-16 acts by interfering with the regulationof the balance between phosphorylation and dephosphorylation of FAKthereby changing the activity of the NKCC1 and other transmembraneproteins. AF-16 counteracts the attempts of these malignant cells toregain the cell volume and simultaneously also their intracellularpressure and dimension.

REFERENCES

-   1. Marks, F., Klingmüller, U., & Müller-Decker, K. Cellular signal    processing. Garland, 2008.-   2. Alberts, B. et al., Molecular biology of the cell. 5th edition,    Garland, 2008-   3. Krauss, G. Biochemistry of signal transduction and regulation.    4th edition. Wiley 2008-   4. Cooper, G. M, & Hausman, R. E. The cell; a molecular approach.    4th edition. Sinauer 2007

1-23. (canceled)
 24. A method for optimizing cellular uptake of apharmaceutical substance and/or formulation, said method comprisingadministering to a subject a pharmaceutical composition comprising anantisecretory factor (AF) protein; a derivative, homologue and/orfragment thereof having equivalent activity; and/or a pharmaceuticallyactive salt thereof.
 25. The method of claim 24 wherein the cellularuptake optimizes delivery of the pharmaceutical substance and/orformulation.
 26. The method of claim 24 wherein said pharmaceuticalsubstance and/or formulation further comprises an anticancer drug, acytostatica, an antimicrobial substance, an antibiotic substance, anantiviral substance or a drug targeting posttraumatic injury,neurodegeneration, a parasite, or an inflammatory condition.
 27. Themethod of claim 24 wherein said pharmaceutical substance and/orformulation treats and/or prevents a tumor and complications thereof.28. The method of claim 24, wherein said antisecretory factor (AF)protein, derivative, homologue, and/or fragment thereof havingequivalent activity, consists of a sequence according to the followingformulaX1-V-C-X2-X3-K-X4-R-X5, wherein X1 is I, amino acids 1-35 of SEQ IDNO:6, or is absent, X2 is H, R or K, X3 is S or L, X4 is T or A, X5 isamino acids 43-46, 43-51, 43-80 or 43-163 of SEQ ID NO:6, or is absent,or a modification thereof not altering the function of the polypeptide.29. The method of claim 24 wherein said pharmaceutical composition is amedical food.
 30. The method of claim 24 wherein said pharmaceuticalcomposition comprises two or more antisecretory factor (AF) proteins.31. The method of claim 24 wherein said pharmaceutical compositionfurther comprises a pharmaceutically acceptable excipient.
 32. Themethod of claim 24 wherein said pharmaceutical composition is formulatedfor intraocular, intranasal, oral, local, subcutaneous and/or systemicadministration.
 33. The method of claim 24 wherein said pharmaceuticalcomposition is formulated for administration as a spray, aerosol,inhaler and/or by a nebulizer.
 34. The method of claim 24, wherein thepharmaceutical composition is formulated for administration systemicallyto the blood at a dose of 0.1 μg to 10 mg per application and kg bodyweight and day.
 35. The method of claim 34, wherein the pharmaceuticalcomposition is formulated for administration systemically to the bloodat a dose of 1-1000 μg per application and kg body weight and day. 36.The method of claim 24, wherein said administration is performed as asingle dose or as multiple daily applications.
 37. The method of claim24, wherein the antisecretory factor (AF) protein, a derivative,homologue, and/or fragment thereof having equivalent activity, isprovided as egg yolk enriched in antisecretory factors.
 38. The methodof claim 24, wherein the antisecretory factor (AF) protein, aderivative, homologue, and/or fragment thereof having equivalentactivity is produced endogenously by a patient after intake of a foodand/or a food for special dietary use that induces the uptake, formationand/or release of an antisecretory factor (AF) protein.
 39. A method fortreating a mammal in need of optimized uptake of a pharmaceuticalsubstance, said method comprising: feeding to the mammal speciallyprocessed cereals (SPC) or egg yolk enriched in antisecretory factors toinduce endogenous production of antisecretory factors for facilitatingoptimized uptake of the pharmaceutical substance, and administering thepharmaceutical substance.
 40. A method for treating a mammal in need ofoptimized uptake of a pharmaceutical substance, said method comprising:administering an antisecretory factor (AF) protein, peptide, derivative,homologue, and/or fragment thereof having equivalent functionalactivity, or a modification thereof not altering the function of thepolypeptide, and/or a pharmaceutically active salt thereof forfacilitating optimized uptake of the pharmaceutical substance, andadministering the pharmaceutical substance.
 41. A pharmaceuticalcomposition comprising: an antisecretory factor (AF) protein, peptide,derivative, homologue, and/or fragment thereof having equivalentfunctional activity, or a modification thereof not altering the functionof the polypeptide, and/or a pharmaceutically active salt thereof, and apharmaceutical substance and/or formulation, wherein said pharmaceuticalsubstance and/or formulation is selected from the group consisting of ananticancer drug, radiation therapy, antibiotic substance, antiviralsubstance and a drug targeting posttraumatic injury, neurodegeneration,a parasite, or an inflammatory condition.
 42. An antisecretory factor(AF) protein, a derivative, homologue, and/or fragment thereof havingequivalent activity, or a modification thereof not altering the functionof the polypeptide, and/or a pharmaceutically active salt thereof whichoptimizes delivery and/or cellular uptake of a pharmaceutical substanceand/or formulation.
 43. A method for designing a diagnostic and/orprognostic tool for monitoring and/or verifying and/or enhancing thetherapeutic control of a malignant tumor in a subject suffering fromcancer, said method comprising: measuring the quantitative occurrence ofphosphorylated FAK in isolated samples of the tumor tissue or isolatedcells from the subject.
 44. A method for improved drug designcomprising: testing the response of cells or tissue, subject totreatment of a substance or a pharmaceutical composition, and estimatingthe influence of antisecretory factors (AF) on said drug response bymeasuring the quantitative occurrence of phosphorylated FAK.
 45. Amethod for screening for and/or evaluating potential antisecretoryfactor (AF) proteins, said method comprising: exposing antisecretoryfactor (AF) proteins, peptides, derivatives, homologues, and/orfragments thereof, having equivalent functional activity, and/or apharmaceutically active salt thereof to a selected substance, andtesting the ability of said exposed AF to regulate FAK at the NKCC1complex by measuring the quantitative occurrence of phosphorylated FAK.46. The method according to claim 46, wherein the exposed AF is testedby high-throughput screening.
 47. A method for evaluating efficacyand/or for verifying functional activity of new or known antisecretoryfactor (AF) proteins, peptides, derivatives, homologues, and/orfragments thereof, and/or a pharmaceutically active salt thereof, saidmethod comprising: testing the ability of said new or known AF toregulate FAK at the NKCC1 complex by measuring the quantitativeoccurrence of phosphorylated FAK.
 48. A method for screening for and/orevaluating potential new antisecretory factors (AF), said methodcomprising: testing the ability of said potential new AF to regulate FAKat the NKCC1 complex by measuring the quantitative occurrence ofphosphorylated FAK.